The overall goal of this project is to uncover molecular determinants for subtype specificity and allosteric functional modulations of N-methyl-D-aspartate (NMDA) receptors. NMDA receptors belong to a family of ionotropic glutamate receptors (iGluRs) involved in the majority of excitatory synaptic transmission in the mammalian brain. NMDA receptors are multimeric ligand-gated ion channels composed of NR1 and NR2 subunits that bind to glycine and L-glutamate at the extracellular domain (ATD/S1S2), respectively. Gating or opening of transmembrane ion channels is mediated by binding of both glycine and L-glutamate to the ligand- binding domain (S1S2) and are allosterically modulated by binding of modulator compounds including phenylethanolamines, polyamines, protons, and Zn2+ to the amino terminal domain (ATD). The functional properties of NMDA receptors differ significantly depending on their subtypes that are defined by four distinct NR2 subunits (A though D). Although several structures have been previously determined for the S1S2 from NR1 and NR2A, the molecular basis for subtype specific ligand-bindings and functions including exceptionally slow deactivation kinetics of the NR2D containing NMDA receptors is unknown due to a lack of structural information on the other NR2 subunits. Furthermore, the general mechanism of activation and inhibition in the NR2 subunits remains an open question because the structure of the NR2 S1S2 in complex with any partial agonist or antagonist has yet to be elucidated. Finally, the molecular mechanism for allosteric modulation mediated by the binding of modulator compounds to the ATD remains elusive due to a complete lack of the ATD structures. Thus, our goal is to obtain the atomic view of the ATD and S1S2 of the NMDA receptors to reveal the molecular mechanism for subtype specificity and allosteric modulation mediated through modulator binding in the ATD. The experimental plan combines x-ray crystallography, electrophysiology, and biochemical techniques. The structural information of the extracellular domain of the NMDA receptors, ligand-binding core (S1S2), and amino terminal domain (ATD), will be complemented by mutagenesis coupled with electrophysiology and biochemical experiments to establish the structure-function relationships of the NMDA receptors. There are two specific aims in this proposal. Aim 1 is to understand the structural mechanism for the subtype specific ligand-bindings, activation, inhibition, and deactivation mediated through NR2 S1S2. Aim 2 is to decipher the molecular mechanism underlying allosteric modulation of the NMDA receptor activity mediated by the binding of allosteric modulators, including Zn2+, ifenprodil, proton, and polyamines, to the NR1 and NR2B ATDs. The NMDA receptors have been a major target for pharmacological studies because they play pivotal roles in brain function and development. Dysfunction of the NMDA receptors is implicated in neurological and mental health related diseases and injuries, including seizure, schizophrenia, Alzheimer's disease, and Parkinson's disease. The results of this research are expected to help design novel compounds that target the ATD and S1S2 with high specificity and potency and with significant therapeutic values.